This competitive renewal will continue our work on understanding the pathophysiology of cryptococcal disease in the central nervous system (CNS) from the yeast's perspective. Cryptococcus neoformans and Cryptococcus gattii represent encapsulated basidiomycetous yeasts that produce a million cases of disease per year and an estimated 600, 000 deaths with present treatment. Cryptococcus is neurotropic causing cryptococcal meningoencephalitis, the life-threatening stage of infection for this disease. Aim 1 will not only define the in vivo transcriptional response of Cryptococcus clinical isolates to human CNS, but it will also identify gene(s) and gene pathway(s) that contribute significantly to the yeasts ability to grow and survive in the CNS. Furthermore, we will characterize these responses based on patient outcome (more or less virulent), genotype, and additional variables to identify conserved genes that may contribute to strain adaptation in the CNS to produce disease. The impact of these genes will be assessed using robust assays that examine their impact on survival in human CSF, virulence determinants, survival in animal models. Aim 2 continues our focused analysis on how core metabolism promotes cryptococcal growth and survival in the CNS by investigating carbon and nitrogen metabolism. This application integrates advanced molecular techniques, insights of the fungus through our prior studies, and close linkage to human disease and outcome. Its goal is to dissect, identify, and validate weak spots in the yeast's stress response in the CNS and thus provides new target (s) for antifungal strategies, which continue to substantially fail in the treatment of this life-threatening fungal infection.